JPH02471A - Production of l-lysine - Google Patents

Abstract

PURPOSE:To increase productivity of L-lysine by culturing microorganism having specific recombinant DNA. CONSTITUTION:A microorganism having a recombinant DNA of a DNA fragment containing gene having activity transforming microorganism belonging to Corynebacterium or Brevibacterium, derived from Corynebacterium mycetoma or Brevibacterium mycetoma and exhibiting sensitivity to S-(2-aminoethyl)- cysteine (e.g., Corynebacterium glutamicum L-15 FERM-P 5946) to S-(2- aminoethyl)-cysteine resistant strain and a vector DNA capable of autonomic replicating in Corynebacterium mycetoma or Brevibacterium mycetoma (e.g., pCG11 ATCC-39022) is inoculated to a medium, cultured with shaking and L- lysine is generated and accumulated in the cultured substance, then L-lysine is gathered from the cultured substance.

Description

[Detailed description of the invention] The present invention relates to a novel method for gene expression.

More specifically, the present invention provides a recombinant product of a DNA fragment containing at least one gene and a vector DNA, and
A transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium using a recombinant DNA in which at least one of the NAs is foreign to the host strain is used as a medium. The present invention relates to a method for expressing a gene, which is characterized by culturing and expressing the trait of the gene.

Recombinant gene technology has been established using Escherichia coli as a host, and to date it has been possible to produce bebutides and vaccines such as somatostatin, insulin, human growth hormone, human interferon-α, human interferon-β, and mouth ulcer vaccine. It has been shown. Although Escherichia coli is considered to be sufficient as a host for the expression of these highly bioactive peptides and vaccines in many cases, higher productivity, extracellular secretion, and glycosylation are required, or the contamination of intracellular toxins is avoided. Therefore, yeast and Bacillus subtilis have also been developed as hosts.

When producing physiologically active substances such as peptides and proteins, it is sufficient to use strains for which recombinant DNA techniques such as those mentioned above have already been established or for which the basics are in place. When using recombinant DNA techniques to improve the industrial productivity of substances such as antibiotics, it is necessary to devise ways to apply the same techniques to each of the production bacteria that have been used conventionally.

Corynebacterium glutamicum was the first microorganism to be used for the industrial production of amino acids. Since then, Coryneform bacteria, including the genus Corynebacterium, have been used to produce glutamic acid, lysine, alanine, histidine, tryptophan, tyrosine, phenylalanine, threonine, and amino acids. Industrial production of amino acids such as leucine, valine, leucine, glutamine, proline, and arginine has been developed, and today most amino acids are produced by microorganisms.

Therefore, the establishment of recombinant DNA techniques in these microorganisms is considered to be extremely important for improving amino acid production in the future.

Recombinant DNA techniques include, for example, (1) fragmentation of DNA containing the target gene with restriction enzymes, (2) linearization by single cleavage of vector DNA with the same restriction enzyme, (3) above (1), ( Recombinant D by annealing by mixing the products of 2) and ligation using DNA ligase
Preparation of NA (4) Introduction of the above recombinant DNA into a host strain (transformation) (5) The steps include selection of a recombinant containing the target gene and purification of the selected clone. The efficiency of creating a recombinant strain obtained in this way can be said to be the product of each of the above steps, so it is important to prepare a means to verify each step, know the efficiency of each step, and improve it. It is not possible to obtain a transformed strain capable of expressing the target gene. Furthermore, even if a transformed strain containing recombinant DNA containing the target gene is obtained in this way, there are various barriers to expressing the gene if the gene is foreign to the host strain. It is known that [“Chemistry and Biology” 18.110-1
18 (197g)) its expression is very difficult to achieve.

Recombinant D using a microorganism belonging to the genus Corynebacterium or Brevibacterium as a host and containing a target gene or vector foreign to the host.
Until now, there has been no known example of expressing the trait of the target gene by introducing NA.

In recombinant DNA techniques using microorganisms belonging to the genus Corynebacterium or Brevibacterium as hosts,
It is necessary to construct a vector system that autonomously replicates in these microorganisms, has a selectable phenotype, and can be used for cloning many genes, and to establish an efficient transformation system. Furthermore, it is necessary to establish a method to eliminate the above-mentioned barriers.

The present inventors previously constructed a plasmid vector that replicates autonomously in microorganisms belonging to the genus Corynebacterium or Brevibacterium and has a selectable phenotype and a suitable loning site, while developing a highly efficient transformation system. developed
9), 56-58187 (Unexamined Japanese Patent Publication No. 5718649)
2), 56-65777 (Unexamined Japanese Patent Publication No. 57-1864
89) ).

Therefore, the present inventors applied the well-known in vitro DNA assembly technique (tl,
Corynebacterium glutamicum strain 122 or its derivatives was ligated using the developed transformation system. When the cells were transformed, the foreign gene was expressed in the host, and the present inventors discovered that it could be used to increase the production of useful substances such as amino acids, leading to the completion of the present invention.

The present invention will be explained in detail below.

The present invention relates to a recombinant DNA that is a recombinant of a DNA fragment containing at least one gene and a vector DNA, and in which at least one of both DNAs is foreign to the host strain.
The present invention provides a method for transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium using A, culturing the resulting transformant in a medium, and expressing the trait of the gene.

DNA fragments containing genes used in the present invention include DNA fragments derived from eukaryotes, prokaryotes, viruses, bacteriophages, or plasmids and containing at least one complete gene.

Examples of genes derived from eukaryotes include genes encoding bebutides such as interferon, insulin, and growth hormone in mammals, particularly humans. Genes derived from prokaryotes include bacteria, especially Escherichia, Corynebacterium, Brevibacterium,
Genes derived from bacterial strains belonging to the genus Bacillus or Staphylococcus, including genes involved in cell metabolism, particularly synthetic activity. Cellular metabolic or synthetic activity refers to the synthesis of amino acids, vitamins, nucleic acids, or antibiotics, as well as the metabolic systems involved in the synthesis. Activity is preferably mentioned.

Furthermore, when the amino acid composition of the target peptide, protein, etc. is known, the corresponding DNA can also be synthesized and used. Examples of DNA synthesis methods include K, 1
takura et LL 5science 1ift
, 1056 (1977).

The vector used in the present invention must be compatible with the host bacterium (c
It must be able to reproduce autonomously. As a specific example, the present inventors have collected pcGl from a microorganism belonging to the genus Corynebacterium, or produced it by inducing the collected material
N Japanese Patent Application No. 57-134500)), pCG2 [Patent Application No. 56-133557 (Japanese Patent Application No. 58-35197)
), pCG4 [Japanese Patent Application No. 56-58186
7-183799) ), ρCE53, pcE54, p
Examples include cGII, pcBlol, and I)Ethrl.

Bacterial strains harboring these plasmids have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology and the American Type Culture Collection under the following deposit numbers.

Plasmid FERM-P ATCCp
CG 1 5865 31808p C
G2 5954 31832p CG
4 5939 31830p CE5
4 39019p CG 11
39022p CB 101
39020pEthr 1
39021 preferably pcGll, 9Cε
54 is used. pcGll was previously disclosed by the present inventors [Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500)
)) Corynebacterium glutamicum 225-57 (ATCC3180g, FER
Plasmid pCG l isolated from M-P5865)
Corynebacterium glutamicum 225-250 (AT
This is a plasmid in which the BamH1 fragment containing the streptomycin and/or spectinomycin resistance (Sm''/5pec'') gene of plasmid pCG4 isolated from plasmid pCG4 (CC31830, FERM-P5939) is ligated using the same cohesive ends of both.

pcGII is a 6.8 Kb plasmid with Bgj! as a single restriction site. II, pst I and S
m”/5 pec”.

pEC54 can be created as follows.

First, pCG2 was transferred to the nursery Corynebacterium glutamicum strain 225-218 (FERM-P5954,
Patent application 1982-13 from cultured bacterial cells of ATCC31832)
3557 (Japanese Unexamined Patent Publication No. 5835197), pGA22 is concentrated and isolated from cultured cells of E. coli harboring it by a commonly used method. Both plasmid DNAs are digested with a restriction enzyme that has one cut point in each molecule, such as Pstr.
After complete digestion and linearization, T4 phage D was used to generate a conjugate molecule in which both DNA molecules were linked with the same cohesive ends protruding from both ends of the plasmid molecule as a single strand.
Apply NA'If gauze. To obtain a recombinant plasmid in which both plasmid molecules are conjugated and linked from this DNA mixture, first, a Corynebacterium or Brevibacterium species selected for single-drug resistance derived from pGA22 is obtained. This is achieved by isolating transformed strains and analyzing the plasmids possessed by these transformed strains.

Transformation with a DNA hybrid can be carried out using a transformation method using protoplasts of Corynebacterium and Brevibacterium species previously disclosed by the present inventors [Patent Application No. 5]
6-58187 (Japanese Unexamined Patent Publication No. 57-186492) and Japanese Patent Application No. 56-65777 (Unexamined Japanese Patent Application No. 57-186489)
) can be implemented. The drug used for selection is p
Among the drug resistance genes derived from G^22, pG82
Tetracycline (TC), chloramphenicol (Cm), or kanamycin (Ni...) corresponding to other resistance genes other than the ampicillin resistance gene, which is inserted and inactivated because it serves as a linking site with the ampicillin resistance gene, may be used. The transformed strain is prepared in a DNA-free system with a concentration of drugs (usually tetracycline 0.4-1.6x/ll111 chloramphus-'-col 2.5-5x/m+ and Kanamycin 100-
Either isolate the reverting colonies on a hypertonic agar medium containing 800 cells/ml) or - scrape them after non-selectively reverting to normal cells on a regeneration medium and transfer this resuspension to recipient bacteria. Concentrations of drugs at which normal cells cannot grow (usually 0.5-4 q/ml of tetracycline, 2-15 g/ml of chloramphenicol and 2-2 q/ml of kanamycin)
51/ml) by isolating colonies growing on an agar medium containing 51/ml). Tetracycline, chloramphenicol or kanamycin resistance (Tc”,
Among the transformed strains selected by Cm" and Km", some have also acquired other drug resistance traits derived from IIGA22.

Plasmid DNA possessed by the thus obtained transformed strain
A was filed by the present inventors in Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No.
7-134500) and Japanese Patent Application No. 56-65777 (Japanese Unexamined Patent Publication No. 57-186489), it can be isolated and purified from cultured bacterial cells, and the DNA fragments produced by digestion with various restriction enzymes can be subjected to agarose gel electrophoresis. The structure can be known by the usual method of analysis.

ρCε5 with a plasmid isolated from one of the transformed strains.
It is 4.

pCε54 is a plasmid with a size of approximately 14.5 Kb.
εcolt L Sal I, Sm as a single restriction site
a I, Xho I, etc., Tc', Cm'',
K gives the phenotype of.

Xho I is located in the Km'' gene, and selection by so-called insertional inactivation (a phenomenon in which expression of the relevant phenotype is prevented by insertion of a DNA fragment) is also possible.

Collection of plasmids from plasmid-carrying strains is described in, for example, Japanese Patent Application No. 56-1810H (Japanese Unexamined Patent Publication No. 57-134500).
, No. 56-58186 (Japanese Unexamined Patent Publication No. 57-183799) and No. 56-133557 (No. 58-35297)
) may be carried out according to the method described in .

Production of a recombinant between a DNA fragment containing a gene and vector DNA can be carried out by making full use of known in vitro recombinant DNA techniques.

In vitro DNA recombination is usually performed by cutting and recombining donor DNA containing the gene of interest and vector DNA. DNA can be easily cut using restriction enzymes. Restriction enzymes used for in vitro recombination recognize and cleave specific base sequences on all double-stranded DNA, regardless of biological species.

The base sequence differs depending on the type of restriction enzyme.

Therefore, by using an appropriate restriction enzyme, the gene of interest can be excised as a single DNA fragment without impairing its expression function. Donor D cleaved by the same restriction enzyme
The end structures of the cut fragments of NA and vector DNA have the same structure; some restriction enzymes give cohesive ends with a protruding single strand, while other restriction enzymes give blunt ends.

As long as both ends are cleaved with the same restriction enzyme, the cut fragments of donor DNA and vector DNA can be ligated using T4 phage DNA ligase.

Even when both DNAs are cut with different restriction enzymes, for example,
The sticky ends can be repaired with DNA polymerase to make double strands, the ends can be blunted, and then ligated, the complementary homopolymers can be added with terminal transferase to create sticky ends, and then ligated, or some kind of restriction can be used. A synthetic oligonucleotide linker containing an enzyme cleavage site can be ligated and then internally cut to create cohesive ends before ligation. By these ligation methods, a recombinant of a DNA fragment containing the gene of interest and a vector DNA fragment is generated.

In addition to the desired recombinant, other recombinants are generated by the ligase reaction, but in order to obtain the desired recombinant, this DN
Corynebacterium or Brevibacterium species are directly transformed using the mixed solution A, and a transformed strain endowed with genetic traits derived from the genetic information of the target gene is selected and isolated, and the cultured bacterial cells are isolated. This can be achieved by extraction and isolation from Instead of directly transforming Corynebacterium or Brevibacterium species, the gene of interest can be transformed into host-vector systems of other microorganisms, such as E. coli.
After that, a recombinant with a vector of a Corynebacterium or Brevibacterium species is produced in vitro, and then a Corynebacterium or Brevibacterium species is transformed in the same manner as above. Recombinants can also be obtained by selectively separating transformed strains.

For recombinant production, the descriptions in the following documents can be widely applied.

S N, Cohen, s, LaL tl, s, Pat
ent 4.237, 224, Genetic Manipulation Experimental Method [edited by Yasutaka Takagi, Kodansha Sun Entifice/Re (1980)
] , Method in Enzymology6
8. Recomb+nant DNA edited
by Ray 11u, ^ academic Press
1979 As the host microorganism of the present invention, any strain belonging to the genus Corynebacterium or Brevibacterium and having the ability to take up DNA may be used. Preferably, the lysozyme-sensitive microorganisms previously disclosed by the present inventors in Japanese Patent Application No. 56-151464 (Japanese Patent Application Laid-open No. 58-56678) are used. Specific examples of bacterial strains include the following strains.

Deposit number F[! R11-P ATCC Corynebacterium glutamicum L-155946
31834 Corynebacterium octatus L-1
03594731866 Previbacterium daylicolumn L-204594831867 Previbacterium lacto 7 amentum L-312594931
868 Transformation of host microorganism with recombinant DNA is 1
This process is carried out in the following steps:) preparation of protoplasts from cultured cells, 2) transformation of protoplasts with recombinant DNA, and 3) regeneration of protoplasts into normal cells and selection of transformed strains. Examples of specific methods are shown below.

l) Preparation of protoplasts from cultured cells Protoplast formation is performed by growing microorganisms under conditions that make them sensitive to the cell wall lytic enzyme lysozyme, and then treating the cultured cells with lysozyme in a hypertonic solution to dissolve and remove the cell wall. Various cell wall synthesis inhibitors are used to convert microorganisms into lysozyme-sensitive cells. For example, microbial cells can be made sensitive to lysozyme by adding penicillin at a concentration that does not inhibit or semi-inhibit growth in the middle of the logarithmic growth phase of microbial culture and allowing the culture to grow for several generations.

The medium used at this time may be any medium that allows microorganisms to grow, such as nutrient medium NB (medium containing 20 g of powdered bouillon and 5 g of yeast extract in pure water II! and adjusted to pH 7.2) or semi-synthetic medium SSM [ glucose 10g
5NH4CIl 4 g, urea 2 g, yeast extract Ig
, KN2P port 4 1 gSK2HPO13g114gC
L・6) 120 0.4 g, FeSO44H2
010mg, MnS mouth, 4-6L mouth Q, 2 mg,
Zn5O<・7HzOo, 9 a+g1CuSO1
・5Hz00.4ff1g.

Na2B<0t40H200,09mg5 <N■<>
*Mo70t<・4Hz80, 04 mg, biotin 3
0 ■, containing 1 mg of thiamine hydrochloride in water 11, pH 7
, 2], etc. are used.

Microorganisms are inoculated into this medium and cultured with shaking.

Measure the absorbance (00) at 660r+m with a colorimeter and adjust the concentration to 0.1-2.0 units/m I in the culture solution at the beginning of the logarithmic growth phase (OD=O, 1-0,4). Add a penicillin such as penicillin G. The culture is continued further, and when the OD increases to 0.3 to 0.5, the cells are harvested and washed with 33M medium. The cells are then cultured in a suitable hypertonic medium, such as PFM medium (S3M 2-fold diluted with 0.4 M SMgCj!2.6H20).
0.01M and adjusted to p) 17.0 to 8.5) or RCG medium [5 g of glucose, 5 g of casein hydrolyzate, 25 g of yeast extract, K2HP0.
3.5g5KLPO41.5g, 14gcL・6)12
0 0.41gFe5 mouth<・7H*0 10mg5 M
n5O*・4~6) 120 2 tagS 1nsO<
・7H200,9mg, CuSO4'5H7Oo,
4 mg, NaJn01401 (zoo, 09 m
g, (N)+4)6M0.024'4)+200.0
4 mgs biotin 30Jig, thiamine hydrochloride 2m
resuspended in a medium containing 1.35 g of disodium succinate in 11 parts of water and adjusted to pH 1.0 to 8.5.

Lysozyme is added to this cell suspension at a final concentration of 0.2-10 mg/ml and reacted at 30-37°C.

Protoplast formation progresses as the reaction time progresses, and its progress can be observed using an optical microscope. The time required for most cells to become protoplasts under the microscope varies depending on the concentration of penicillin added during cell culture and the concentration of lysozyme used, but is 3 to 24 hours under the above conditions.

The generated protoplasts undergo rupture array under hypotonic conditions, so
The degree of protoplast formation can be indirectly determined by the degree of remaining normal cells that survive under hypotonic conditions. Normally, the residual level of normal cells can be suppressed to about 10-4 of normal cells treated with lysozyme.

In this way, Jj! The illed protoplasts have the ability to form colonies (regenerate) on an appropriate hypertonic agar medium. This agar medium may be a nutrient medium, a semi-synthetic medium, or a synthetic medium supplemented with several types of amino acids with a concentration of 0.3 to 0.
8M disodium succinate and 0.5-6% polyvinylpyrrolidone (110.000 or 40.00 mol.
0> is preferably used.

Usually, semi-synthetic medium RCGP medium (RCG medium with 3% polyvinylpyrrolidone (molecular weight 10.000) and 1.4
% of agar, pH 7.2) can be used. Cultivation is preferably carried out at 25-35°C.

The number of culture days required for the appearance of regenerated colonies varies depending on the strain, but it takes 10 to 14 days for the colonies to reach a size that can be harvested.

Regeneration of protoplasts in RCGP medium varies depending on the bacterial species, the concentration of peninrin added during the culture, and the concentration of lysozyme treatment.
The efficiency is between 2 and 10-'.

2) Transformation of protoplasts with recombinant DNA Uptake of recombinant DNA into protoplasts involves mixing protoplasts and recombinant DNA in a hypertonic solution that allows cells to maintain a protoplast state, and adding a DNA uptake-mediated effect to this. This is carried out by adding a divalent metal cation to polyethylene glycol (PEG, average molecular weight 1.540 to 6.000) or polyvinyl alcohol <pvA, M synthetic leather 500 to 1,500>. Stabilizers that provide hypertonic conditions may be those commonly used to maintain protoplasts of microorganisms, such as sucrose or disodium succinate. P
Usable concentration ranges for EG and pvA are 5-60% and 1-20% final concentration, respectively. Divalent metal cations include Ca+゛, Mg'', Mn'', and 9 with a maximum concentration of 1-100d.
a゛+5r-- etc. are effective and can be used alone or in combination. The temperature of the treatment is preferably 0 to 25°C.

3) Regeneration of protoplasts into normal cells and selection of transformed strains! To regenerate protoplasts transformed with lI recombinant DNA, protoplasts are spread on a hypertonic agar medium (for example, RCGP medium) containing disodium succinate and polypyrrolidone, and normal cells are regenerated. This is done by culturing at a temperature that allows for growth, generally from 25 to 35°C. The transformed strain is donor D
It can be obtained by selecting for the trait that a gene derived from NA imparts to the fungus. Selection based on the acquisition of characteristic traits may be carried out simultaneously with regeneration on a hypertonic agar medium, or it may be carried out on a normal hypotonic agar medium after non-selective regeneration and then the regenerated normal cells are collected. good.

When using a lysozyme-sensitive strain shown as a specifically preferred host strain in the present invention, the transformation is carried out in the steps described above except for directly treating cells that have been cultured and grown without penicillin treatment in step 1) above. 1) to 3) It can be done in the strike direction. When using lysozyme-sensitive microorganisms, the transformed strain is 10-4 to 10-4 per regenerating microorganism.
Obtained at a high frequency of 10-6.

The transformed strain can express the characteristics of the introduced recombinant DNA by culturing it in a conventional nutrient medium.

If the recombinant DNA has properties derived from genetic DNA or vector DNA, the culture medium may be supplemented with a drug depending on the properties.

Collection of useful substances such as amino acids produced by the method for expressing the characteristics of the present invention is carried out by a conventional method of collecting these substances from a fermentation liquid.

The present invention has made it possible to increase or provide new productivity for amino acids, nucleic acids, vitamins, antibiotics, enzymes, peptides, and proteins in microorganisms of the genus Corynebacterium and Brevibacterium. It has also become possible to improve the production method by enhancing the metabolic activity of microorganisms, increasing their substrate utilization, providing new metabolic activity, and providing new substrate utilization.

Furthermore, in the present invention? ! fy:1. has successfully expressed heterologous genes or foreign recombinant DNA in microorganisms of the genus Corynebacterium or Brevibacterium. Specifically, the threonine operon of Escherichia coli, the phosphoenolpyruvate carboxylase (PPC) gene, and the gene pHB110 expressed in Bacillus subtilis and Staphylococcus (Kegg
ins K,! J., et al., Proc.
Natl^cad, Sci,, U, S, A, Li
The kanamain resistance gene of Corynebacterium glutamicum, the gene involved in lysine biosynthesis of Corynebacterium glutamicum, and the anthranilate synthase gene of Brevibacterium flavum were expressed in Corynebacterium bacteria.

All of the exemplified genes were simply introduced in the form of a ligation to a Corynebacterium glutamicum plasmid, and no special manipulation was performed to express them in Corynebacterium glutamicum. In addition, D containing the gene
No matter which direction the NA fragment is ligated to the Corynebacterium glutamicum plasmid, it will be expressed in Corynebacterium glutamicum. - It is clear that it has the ability to accurately recognize the starting point of translation and carry out transcription and translation. Considering that, as is well known, all genes contain similar sites at the base sequence level that are necessary for accurate initiation of transcription and translation, Corynebacterium glutamicum is an example. It is easily inferred that the transcription/translation initiation sites of genes other than those of genes can also be recognized and expressed.

Although the so-called glutamate-producing bacteria that have a high glutamate production ability have the same main mycological properties, each researcher has assigned various enclaves and genus names due to their industrial importance. Even the genus Corynebacterium and Brevibacterium are diverse. However, it has been pointed out that these bacterial groups are the same bacterial species because the amino acid composition of their cell walls and the base composition of their DNA are uniform. Furthermore, it has recently been revealed that there is more than 70-80% DNA homology between these bacterial species, and it is clear that they are very closely related microorganisms.Komatsu, Y.: Report of
theFermentative Re5earc
h In5titude, No, 55. 1 (1
980) and 5uzuki, ni, aneko,
T, and Komagata.

: Int, J, 5yst, Bac
teriol, 31. 13H1981)]. Since the hosts that can be used for recombinant DNA experiments are regulated herein, the usefulness of the present invention is limited to Corynebacterium spp.
Although a derivative strain of G. glutamicum shi-22 was shown as a host, based on the above facts, it can be easily inferred that it can be applied as it is to all glutamic acid producing bacteria. Recombinant DN
In order for A to be stably retained and expressed in these bacterial species, slight differences in host bacterial properties such as DNA homology are not a problem, and these bacterial species must be able to autonomously replicate the plasmid and express the introduced gene. It suffices if it has a function that enables it. However, the fact that these bacterial species share both of these functions was previously disclosed by the present inventors [Patent Application No.
Plasmid ρCG4, which is isolated from Corynebacterium glutamicum 225-250 and has a streptomycin and/or spectinomycin resistance gene, can be used to isolate Corynebacterium and Brevibacterium species, etc. It can also be replicated in glutamic acid-producing bacteria, and its resistance gene is expressed [Patent application No. 56-5111187
(Japanese Unexamined Patent Publication No. 57-186492)). Therefore, host bacteria to which the present invention can be applied include not only Corynebacterium glutamicum but also all glutamic acid-producing bacteria including Corynebacterium and Brevibacterium species.

Examples of the present invention are shown below.

Example 1. Cloning of the gene involved in lysine biosynthesis of the lysine-producing bacterium Corynebacterium glutamicum^TCC21543 in Corynebacterium glutamicum and production of lysine by Corynebacterium glutamicum using the expression of the gene: (1) Corynebacterium glutamicum ATC [
:21543 chromosomal DNA and preparation of vector pcG11: Corynebacterium glutamicum ATCC130
The chromosomal DNA of Corynebacterium glutamicum^TCC21543, a lysine-producing mutant strain derived from 32 and resistant to the lysine analog S-(2-aminoethyl)-cysteine (hereinafter abbreviated as ΔEC), was prepared as follows. Extract and isolate.

40 Qml semi-synthetic medium SSMC glucose 20g1 (
NH4) 2S0410 g, urea 3 g, yeast extract I
g, K+(2P04 1 g, MgC1x-68z
0 0.4 g, Fe5D<・7Hz010mg, l
1nSOs'4-6H20Q, 2 mg, ZnSO4'
7) 1200.9mg, Cu5On'5Hz00.4
mgSNa2B40t'1OH20o, 09'gs
(Ni14)6Moth24・4L00.04 mg
- Add 100 g of threonine to a medium containing 30 g of biotin and 1 z of thiamine hydrochloride in 1 z of water and adjusting the pH to 7.2.
Inoculate the seed culture into the supplemented medium to give 3.
Culture with shaking at 0°C. The absorbance (00) at 660 nm is measured using a Tokyo Photoden colorimeter, and when the OD reaches 0.2, penicillin G is added to the culture solution at a concentration of 0.5 h/ml. Further culture was continued until the OD was approximately 0.
Grow until 6 years old.

Collect bacterial cells from the culture solution and add TBS buffer (0.03M tris(hydroxymethyl)aminomethane (hereinafter abbreviated as Tris), 0.005!4E (lTA, 0.051N)
aCj! After washing with lysozyme solution (pH 8, 0)
5%5% stain 0.1&N1aCj! , 0.05M) Lis, 0.8mg 7ml lysozyme: pH 8.0 or less, the same) Suspend in 1Qml and react at 37°C for 4 hours. From the collected bacteria, Saito et al.'s method (Saito, ) I,
et al: Biochim.

Biophys, ^cta, 72, 619 (196
3) Isolate high molecular weight chromosomal DNA according to :l.

On the other hand, pcGll used as a vector plasmid is
Corynebacterium glutamicum strain 22 derivative strain L^103 carrying pcGll^103/pCG 1
1 (ATCC 39022) as follows.

Culture with shaking at 30° C. in 4QQml NB medium (medium containing 20 g of powdered bouillon and 5 g of yeast extract in water lI! and adjusted to p) 17.2 until the OD reaches approximately 0.7. The bacterial cells are collected, washed with TESll solution, suspended in lysozyme solution IQml, and reacted at 37°C for 2 hours. 5M NaCj! to the reaction solution. 2.4ml, 0.5MEDT
A (pHI 3.5) 0.6+nl, solution 4.4 consisting of 4% sodium lauryl sulfate and 0.1M NaCR
ml in order, mix gently and place in ice water for 15 ml.
Give it time.

Transfer the entire lysate to a centrifuge tube and incubate at 4°C for 60 min.
Centrifuge at 0 x g and collect the supernatant.

Add polyethylene glycol (PBG) 6.000 (manufactured by Hanui Chemical Co., Ltd.) equivalent to 10% by weight to this, mix gently to dissolve, and then place in ice water. 10 hours later 1
．． 5f) Collect pellets by centrifugation at OX g for 10 minutes. Gently redissolve the pellet by adding 5 ml of TES buffer followed by 1.5 mg/ml ethidium bromide.2. Add Oml and add cesium chloride to this and gently dissolve to adjust the density to 1.580. Add this solution to 1
Ultracentrifuge at 05.000 x g for 48 hours at 18°C. The circular DNA closed with covalent bonds by this density gradient centrifugation is found as a high-density band in the lower part of the centrifuge tube by irradiation with ultraviolet rays. The pcGll DNA is isolated by extracting this band from the side of the centrifuge tube with a syringe. Next, the fractionated solution was mixed with an equal volume of isopropyl alcohol solution [volume 1 d
Fraction: 90% isopropyl alcohol, 10% TES buffer (this mixture contains a saturated dissolved amount of cesium chloride)]
The ethidium bromide is extracted and removed by treatment five times with 5 times, followed by dialysis against TBS buffer.

(2) Corynebacterium glutamicum ATCC
Cloning of genes involved in lysine biosynthesis of 21543 Reaction solution for restriction enzyme Bgj'n containing the pcG11 plasmid DNAΔ3JLg prepared above (1 (bnM)) Liss-HCl, 7mM IJgcf2.60d NaCL
7d 2-mercaptoethanol, pH 7.5) 60J
Adding 6 units of BgfU (manufactured by Takara Shuzo Co., Ltd.) to I11,
After reacting at 37°C for 60 minutes, the reaction is stopped by heating at 65°C for 10 minutes. On the other hand, Corynebacterium glutamicum A
Restriction enzyme 3amHI reaction solution (10mM) containing 8ug of TCC21543 chromosomal DNA, Lis-HCl, ? +nMM
gCL, 100mM NaCj! , 2mM 2-1 lkabutoethanol, 0.01% bovine serum albumin, pH
8,0) Add 4 units of 3dmHI to 140 Jttt, react at 37°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction.

Mix the raindrops and add T4'J gauze buffer (Tris-HCl 660mM, MgC1'266111M, dithiothreitol 1100In, pH 7.6 > 40m,
ATP (5d) 40111, T4 ligase (manufactured by Takara Shuzo Co., Ltd., 1 unit/1dl) 0.3td and H2O12hj
! and react at 12°C for 16 hours. The mixture is extracted twice with 400 ρ of phenol saturated with TES buffer and dialyzed against TES aqueous solution to remove the phenol.

This ligase reaction mixture was combined with ABC-sensitive LP derived from Corynebacterium glutamicum upper-22 strain.
4 strains are used for transformation.

Transformation is performed using protoplasts of the LP4 strain.

A seed culture of LP4 strain is inoculated into NB medium and cultured with shaking at 30°C. When the ODo reached 6, the cells were collected and the cells were subjected to RC.
GP medium [5 g glucose, 5 g casamino acids, 2.5 g yeast extract, 2 HP ports.

3.5g, H2PO, 1.5g, l! gCl2
・6t+to o, 41g, FeSO4-71120
10mg5 MIISO4・4~6H202ff1g.

2r+5O1-7H200,9mg, (N)I,),!
Jot02<・4H200,04US biotin 30■,
Thiamine hydrochloride 2mg, disodium succinate 135
g, polyvinylpyrrolidone (molecules! 110,000)
1 mg/ml in a medium containing 30 g in 1 i' of water; about 109 thin capsules/m in a solution containing Norizozyme (pH 7.6)
The suspension was transferred to an L-shaped test tube and subjected to gentle shaking reaction at 30°C for 5 hours to form protoplasts.

Take 5+ nl of this protoplast bacterial solution Q into a small test tube, centrifuge at 2500xg for 5 minutes, and add TSMC buffer (10
mM magnesium chloride, 30 mM calcium chloride, 50
ITIM Tris, 400mM sucrose, pH 7,5) 1m
After resuspending in 1ml of TSMC buffer Q and washing by centrifugation, add 1ml of TSMC buffer Q.
Resuspend in Add to this bacterial solution a 1:1 mixture of 2 times higher concentration of TSMC buffer solution and the above ligase reaction DNA mixture.
m and mix, then 20% P in TSMC buffer.
Add 3 ml of solution Q containing EG6.000 and mix. After 3 minutes, add 2 ml of RCGP medium (pH 7,2), centrifuge at 2.500 x g for 5 minutes to remove the supernatant, suspend the precipitated protoplasts in 1 ml of RCGP medium, and then add 2 ml of Q. RCGP agar containing spectinomycin 400 x/ml (RC
It is plated on GP medium containing 1.4% agar, p) 17.2), and cultured at 30°C for 7 days.

All bacteria grown on the agar medium are collected, washed with physiological saline, and then suspended in 1 ml of physiological saline.

This bacterial solution contains 2 mg/ml of threonine and 2 mg/+ 80C.
Minimal agar medium Ml (glucose 10 g
.

NH,) I2PO 11 g, KCl 0.2 g,
Mg5O<・7H200, 2g, FeSO4'7H
J 10mg, ynsO, -4~68zOO, 2mg,
2nSO<4) 1200.9 mg, CuSO4・
5HJ o, 4 mg1Na2840i'1OH20
0.09mgs (NH4)8MO7024'4H20
0.04mg, biotin 50■, p-aminobenzoic acid 2
, 5 mg, thiamine hydrochloride 1+++g, agar 16g
was reapplied onto a medium containing water 11 and adjusted to pH 7.2 and cultured at 30°C for 3 days. Strains resistant to AEC, spectinomycin, and streptomycin are obtained from the colonies that appear.

The plasmid possessed by these transformed strains is the above-mentioned p
It is isolated in the same manner as cGll was isolated. Using 1 gg of these plasmid DNAs, after complete digestion with the restriction enzyme EcoRI, which has a cleavage site on pcGll, it was analyzed by agarose gel electrophoresis.
was identified. Molecule 1 contains the restriction enzyme Hindl[l] of lambda phage DNA that was run simultaneously on the same agarose gel.
The calculation is based on a standard curve drawn by the migration distance of each fragment of known molecular weight produced by digestion. The plasmid pAec 5 obtained from one of the transformed strains is a molecule! l 10.7
Bg of pcGll in Wb! (2) This is a recombinant plasmid in which the DNA fragment 3.9 and b is inserted into the cleavage site.

The protoplasts of the LP4 strain were transformed using the pAec5 DNA in the same manner as above, and the transformed strain selected for resistance to subectinophicin was simultaneously endowed with εC resistance, as determined by the cleavage pattern of εcoRI. They carry the same plasmid. That is, Corynebacterium glutamicum ATCC21543 in pAec 5
It is clear that the gene governing the ABC resistance trait has been cloned. pAec 5 stock strain is Corynebacterium glutaITlic in American type culture +-': IL/section.
It has been deposited as um K17^TCC39032.

(3) Production of lysine by 11Aec5 strain LP4 strain derived from Corynebacterium glutamicum strain 122 possessing ρ^ec5 strain (ATCC39032
) and non-possessed strains will be tested for lysine production. One platinum loop of bacteria grown on NB agar medium was added to 5mM production medium PI.
(100g glucose, (NH,)2SO424,5
g, H2PO41g, Mg5O<・7H200,
4g.

Fe5Oa'7HzOlo+ag, unsos-4-
Medium containing 6t + 10mg of biotin, 50g of biotin, 200g of thiamine hydrochloride, 50hg of calcium pantothenate, 500g of nicotinic acid, 10g of soybean hydrolyzate, and 30g of carbonic acid in 1p of water and adjusted to pH 7.2]
The cells were inoculated into test tubes containing the following, and cultured with shaking at 30°C for 75 hours. After culturing, the L-lysine production rate in the medium was measured by a colorimetric method using an acidic-copper ninhydrin reaction.
Shown in the table.

1st Table P-4 Example 2. Cloning of genes involved in threonine biosynthesis in Escherichia coli and production of threonine by Corynebacterium glutamicum using expression of the genes: Introduction: Cloning is carried out using an E. coli host vector system. pG^22 used as a vector was prepared by the method used by Ahn et al. who created this plasmid [^n, G, a
t at: J, Bacte mouth of,
, Lm 400 (1979)], the E. coli carrying this plasmid was isolated from cultured cells of 112 substrains. High-molecular chromosomal DNA, which serves as donor DNA, was extracted from cultured E. coli strains of 12 strains (ATCI"23740) using Smith's phenol extraction method (Smith, M, G
, : ! Jethod inEnzy-molog
Y, 12. part A, 545 (1967)
).

Restriction enzyme 11+ndl1 reaction solution containing 44g of pc^22 plasmid DNA (10mM) Lis-HCl, 7m
M MgCIt 2゜60m! I NaCIl, pl
(7,5) Add 0.4 units of Hindu (manufactured by Takara Shuzo Co., Ltd., 6 units/waste) to 60 m and react at 37°C for 30 minutes.
The reaction is stopped by heating at 5° C. for 10 minutes. There are two HindUI cleavage sites in pGA22, and as a result of agarose gel electrophoresis of a sample digested with old nd[[ under the same conditions, it is confirmed that the pGA22 has been cleaved into - fragments. Separately, the anti-Va enzyme containing chromosome DN^8Itg) 1
jnd1 reaction solution] Add 4 units of old ndm to 40 scraps and add 3
After reacting at 7°C for 60 minutes, heat at 65°C for 1 minute to stop the reaction.

Mix the raindrops and add 40 μISAT of Tl gauze buffer.
Add P(5mM>40m, 0.3g of T4 ligase and)+20t20m and react for 16 hours at 12°C.

This mixture was mixed with phenol 400 saturated with TBS buffer.
Extract twice with ρ and dialyze against TBS buffer to remove phenol.

This ligase reaction mixture was applied to E. coli strain 112 substrain GT-.
3 [J, Bacteriol, 117. 13
3-143 (1974)) (homoserine and diaminopimelic acid auxotrophy).

Competent cells of the GT-3 strain (a strain with DNA uptake ability) were obtained using the method of Dagert et al.
M,, et M on: Gene, L 23 (197
9) Prepare as follows. That is, a culture medium supplemented with diaminopimelic acid so that the concentration of I
．． (medium adjusted to 2), inoculated into 50 III, and
Cultivate at 37°C until the temperature reaches 0.6. Dilute the culture solution with ice water
Cool for 0 minutes and collect bacteria by centrifugation. Resuspend in 20 ITll of chilled 0,1M calcium chloride and place at 0°C for 20 minutes.

Resentrifuge the cells and add O,lM Calcilam chloride 0.5M
1 and leave at 0°C for 18 hours.

200 m of the above ligase reaction mixture was added to 400 pieces of calcium chloride-treated bacterial suspension, mixed, kept at 0°C for 10 minutes, and then heated at 37°C for 5 minutes. Next, 9 ml of L medium is added and cultured with shaking at 37°C for 2 hours. After centrifugal washing twice with physiological saline, transfer to M9 minimal agar medium (Grape W12 gSNH4C) supplemented with kanamycin equivalent to 12.5x/ml.
j! 1g, Na, 1IPO46g.

K112PO43gSMgSO4-7N200.1g
, CaCj! a.2II, 0 15 mg, 4+ ng of thiamine hydrochloride, and 15 g of agar in water IN (medium adjusted to pH 7.2) and cultured at 37° C. for 3 days. The only horn colony that appeared was ampicillin 25 Jtg/mL and chloramphenicol 25 N/mL.
Alternatively, mushrooms containing 25 g/ml of kanamycin have been confirmed to grow on agar media.

From the cultured cells of this transformed strain, pG^22 was obtained in the above (1).
Plasmid DNA was isolated using the same method used to isolate
Do n. This plasmid DNA was analyzed by restriction enzyme digestion and agarose gel electrophoresis. As a result, pGH
It has the structure shown as 2. The DNA fragment inserted into ρG^22 is a previously cloned E. coli operon-containing DNA fragment [:Co55art, P, et
al: ! olec, Gen.

Genet, JJ439 (1979)], pGH2 is confirmed to contain a threonine operon.

Next, a recombinant of pcGll and pGH2 is created. First, pCG l l and pGH2 are each Bgj! II, and Dam) II under appropriate conditions. Mix the digested material containing each plasmid ON^2■, total volume 200g
For T4 ligase buffer 40#, ATP (
5+nM) 40m, T4 ligase 0.2n and it,
o Add 120 idl and react at 12°C for 16 hours.

This mixture was mixed with phenol 400 saturated with TBS buffer.
The phenol is removed by two head extractions and dialysis against TESI buffer. Subsequently, a 1:1 mixture 1004 of twice as concentrated TSMC buffer and the ligase reaction mixture is provided D.
Corynebacterium glutamicum A201 strain (L^
After transforming the protoplasts of the 103 derived strain (homoserine, leucine auxotroph), they are spread on an RCGP agar medium and cultured at 30° C. for 6 days to regenerate and proliferate. The bacteria grown on the entire surface of the agar medium are collected, centrifuged and washed with physiological saline, then reapplied onto a minimal agar medium MI supplemented with 50 u/an l of leucine, and cultured at 30°C for 3 days. Kanamycin 12.5 μ/ml from the colonies that appeared
Alternatively, a strain can be obtained that can grow on NO agar medium containing 100 g/rn I of spectinomycin.

Plasmids are isolated from these transformed strains by ethidium bromide and cesium chloride density gradient centrifugation as described in Example 1 (1).

Using these plasmid DNAs of 0.5μ, the DNA fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes were analyzed by agarose gel electrophoresis, and the molecular weight and each restriction in the plasmid molecule were analyzed. Identify the enzyme cleavage site. -p the plasmid obtained from the strain
It was named Ethr l. Restriction enzymes Pstl, εco
The structure characterized by the RI and Xho I cleavage sites is shown in FIG. pBthr 1 is p to pcGll
It was found to have a structure containing a GH2 threonine operon and a 3 nm HI fragment.

As a result of re-transforming Corynebacterium glutamicum LA103 strain using pEthr l DNA in the same manner as above, homoserine non-auxotrophy and kanamycin and spectinomycin resistance traits were introduced in combination, and these transformed strains , carries a plasmid identical to pEthr 1 characterized by various restriction enzyme cleavage modes. LA1 due to deletion of homoserine dehydrogenase
The reason why the homoserine auxotrophy of strain 03 is restored to homoserine non-auxotrophy by pEthrl is due to the expression of homoserine dehydrogenase located on the E. coli threonine operon.

(2) Creation of pEthr 1-bearing strain Corynebacterium glutamicum L^-10, a threonine-producing bacterium derived from 122 strains of Corynebacterium glutamicum
6 (methionine auxotrophy, AEC resistance, α-amino-β-
The pEthr 1 strain (resistant to hydroxyvaleric acid) is L
A-106 protoplasts are transformed with pEthr].

Protoplasts were obtained by culturing the LA-106 strain in a semi-synthetic medium SSM supplemented with methionine equivalent to 100 ttg/ml, and growing the cells to an OD of approximately 0.6 using the same process as in Example 1 (2). Prepared by processing.

Transformation was carried out in the same manner as in Example 1 (2), and transformed strains were obtained by selection on an RCGP agar medium containing 400 μm/mf of subectinomycin.

The pEthrl-carrying strain is Corynebacter+u in the American Type Culture Collection.
It has been deposited at mglutam+cum as 19^TCC39034.

(3) Production of threonine by a strain carrying pEthr l A threonine production test is carried out between the LA-106 strain obtained as described above and a strain carrying pEthr l (^TCC39 (134)) and a strain not carrying pEthr l. Produced on NB agar medium 1 bacteria
5ml of production medium P2 (glucose 100g)
, (NH,) 2S0420 g , H2PO40,
5g.

Kal (PO40.5g, 11g5O*・7H201
g%FeSOa-711z010mg, Mn5O<・
4-6Hz0 10mL biotin 100g, carbonate 20g 1 methionine 100mg in water il p)
17. Inoculate a test tube containing a culture medium adjusted to 2 for 30 minutes.
Incubate with shaking at ℃ for 75 hours.

After culturing, the culture P solution was subjected to vapor chromatography to develop ninhydrin color, and L-threonine production (2) was measured by colorimetry. The results are shown in Table 2.

Table 2 LA-106 6.1 Example 3 Production of glutamic acid by Corynebacterium glutamicum storing a recombinant plasmid containing the E. coli phosphoenolpyruvate carboxylase (PPC) gene: (1) E. coli PPC DNA containing the gene (this gene is a gene involved in glutamic acid biosynthesis, which is known to convert Glu- to Glu'' in E. coli)
Cloning of the fragment and introduction into Corynebacterium glutamicum: Cloning is performed in the E. coli host-vector system.

p8R322 used as a vector was injected into Escherichia coli using the same method used to prepare pcA22 in Example 1 (1).
Isolate from cultured bacterial cells of substrains. The high molecular weight chromosomal DNA used as the donor DNA was injected into Escherichia coli strain 112 (+) in Example 2 (+).
ATCC 23740) is used.

ρBR322 plasmid DNA 3g and chromosomal DNA
Reaction solution for restriction enzyme MSa containing 9■ (lQn+AI)
Liss hydrochloric acid, 7m1J MgCj! 7.100mM N
aCj! .

7ml 2-mercaptoethanol, 0.01% bovine serum albumin, pH 7.5) IO units of 5ale (manufactured by Takara Shuzo Co., Ltd.) were added to 200n, reacted at 37°C for 60 minutes, and then heated at 65°C for 10 minutes. Stop the reaction.

Add T41J gauze buffer 40〃, A
T P (5m!J) 40tdl, T4 ligase 0.
4Al' and 120 d of water are added and reacted at 12° C. for 16 hours. The mixture is extracted twice with phenol 400β saturated with TES buffer and dialyzed against TES buffer to remove phenol.

This ligase reaction mixture was injected into E. coli and 12 substrains PPC.
2 (Glansdorff, N., Genetic
s, Tan, 167 (1965)) (arg-, thr,
1eu-, his-, Th1-, PPC-.

ST'). Competent cells of strain PPC2 are cultured in L medium supplemented with 2 mg/ml glutamic acid, and prepared in the same manner as in Example 2 (1) to obtain competent cells of strain GT-3. Transformation is carried out in the same manner as in Example 2(1) using 200 g of the ligase reaction mixture. Add 9 ml of L medium and culture with shaking at 37° C. for 2 hours to express the expression. After centrifugal washing twice with physiological saline, the mixture was applied to an M9 minimal agar medium supplemented with 50 μ/ml each of arginine, threonine, leucine, and histidine.
Culture at 37°C for 3 days. The colonies that appeared were treated with ampicillin 2511g/ml or tetracytalline 25g/ml.
Replica on agar medium containing g/mI and incubate at 37°C.
Culture for 24 hours and select those that are ampicillin resistant and tetracytalline sensitive.

Plasmid DNA is isolated from the cultured cells of these transformed strains in the same manner as described above. As a result of analyzing the plasmid pPCt obtained from one of the transformed strains by restriction enzyme digestion and agarose gel electrophoresis, it was found that the Sal of pBR322 was
It turned out to be a recombinant plasmid with a genome size of 8.8 Kb in which the DNA fragment 4.4 and b was inserted into the l cleavage site.

This ppc 1 plasmid was used to transform PPC2 strain in the same manner as above, and all of the transformed strains selected for ampicillin resistance were non-glutamic acid auxotrophic and had the same structure as ppc I, which was characterized by the restriction enzyme cleavage mode. It has a plasmid of This indicates that the E. coli PPC gene has been cloned onto the pPCI plasmid.

The cloned PPC gene was transferred to Corynebacterium
To introduce pcGll and PPC1 into Glutamicum
A recombinant is prepared in host E. coli.

Reaction buffer for restriction enzyme pstI (20d) containing 2 μ each of pcGll and ppc 1 plasmid DNA
IJS hydrochloric acid, 10mM! JgCL, 50m14 (N
L) isL, 0.01% bovine serum albumin, pH 7
, 5) Add 4 units of PstI (manufactured by Takara Shuzo Co., Ltd.) to 200 m, react at 30°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. To this reaction mixture, add 40 tl of slowing solution for T4 ligase and 401 d of ATP (5 ml & l).
! , T4 ligase 0.2JJ! 1 and 120 scraps of water were added, and the mixture was allowed to react at 12°C for 16 hours. After phenol extraction in the same manner as above, phenol is removed by dialysis. This ligase reaction mixture 100IIJI was used to transform PPC2 strain in the same manner as above.

Isolate the plasmid from the resulting colony using the method described above,
Its size was examined by agarose gel electrophoresis. A plasmid with a size of about 15 to 16 Kb was selected, and the PPC2 strain was transformed again with the plasmid to confirm the presence of the PPC gene. As a result of analysis of the plasmid pEppc 1 obtained from one of the previously transformed strains by restriction enzyme digestion and agarose gel electrophoresis, it was found that the genome size was 1, in which pCG 11 and pPcl were conjugated and linked at their Pst I cleavage sites.
It was clearly shown to be a 5.6 Kb recombinant plasmid. Using the pEppc l plasmid DNA thus prepared in Escherichia coli, the LP4 strain derived from Corynebacterium glutamicum 122 strain is transformed.

Transformation was carried out in the same manner as in Example 1 (2), and the transformed strain was R [GP
Obtained from colonies growing on agar medium. Plasmids isolated from these transformed strains were digested with restriction enzymes 5ail or Pst1 alone or both.
By performing heavy digestion and analyzing by agarose gel electrophoresis, it is confirmed that pEppcl is present.

pEppc l strain is Corynebacterium in American type culture: IL/culture.
ruimglutamicum K-18^TCC39
It has been deposited as 033.

(2) Production of glutamic acid by strain carrying pEppc 1: LP4 strain derived from Corynebacterium glutamicum strain 122 carrying strain pEppc 1 (^TC
C39033) and a non-possessing strain were tested for glutamic acid production. Gather up the leaves grown on the NB agar medium,
After washing with saline, 5mM production medium P3 (glucose 50g, (N)14) 2504 3 g 1 urea 3
g, KHzPO40,5g-, 2HPO40,
5g.

! , 1g5O44) IJ o, 5g, Fe5O
s4H2010mg5Mn5On・4-6L010mg
, biotin 3Iig, thiamine hydrochloride 500Jig,
Contains phenol left IQmg in pure water 11, pH 7,
The cells were inoculated into a test tube containing a medium adjusted to 2) and cultured with shaking at 30°C. During culture, 20% urea solution was added to 0.21111
Each sample was added three times and cultured for 40 hours. After culturing, the culture P solution was subjected to paper chromatography, and after coloring with ninhydrin, the amount of L-glutamic acid produced was measured by colorimetry 1.

The results are shown in Table 3.

Table 3 P-4 10.1 LP-4/pEppc 1 15.8 Example 4. Brevibacterium flavum ATCC140
Cloning and expression of 67 anthranilate synthase genes in Corynebacterium glutamicum: Chromosomal DNA of Brevibacterium flavum ATCC14067 is prepared in the same manner as in Example 1 (1).

pCε53 to be used as a vector is isolated from cultured cells of Corynebacterium glutamicum strain L~22 in the same manner as pcGll was isolated in Example 1 (1). pCε53 is a plasmid pcGl of Corynebacterium glutamicum that was previously disclosed by the present inventors.
(Patent application No. 56-18101 (Unexamined patent application No. 57-13450)
0)) and E. coli plasmid pGA22 [A, 6
．． Low” Upper: J, Bacteriol, 140.40
0 (1979)] are conjugated together. For more information, see Bgl, which is the only place on IICGI! U
Bam1 located at two sites, one on IFraB and on pGA22.
Among the I cleavage sites, the BamHI cleavage site that is not within the tetracycline resistance gene is ligated using the same cohesive ends of both restriction enzymes.

pCε53 has a selection marker such as the kanamycin resistance gene derived from pGA22, and has one cleavage site for the restriction enzyme Sal[.

Restriction enzyme 5al1 reaction solution 2 containing 3 ug of pcIE53 plasmid DNA and 9 g of chromosomal DNA prepared above
00 JLl was added with 10 units of Sal[, and after reacting at 37°C for 60 minutes, the reaction was stopped by heating at 65°C for 10 minutes. Add 40% of T41J gauze buffer to this mixed digest.
〃, ATP (5mM) 40Ji1, T4 ligase 0.44 and H,0120m were added and incubated at 12°C for 16
Allow time to react. The mixture is extracted with 400 m of phenol saturated with TES buffer and dialyzed against TES buffer to remove the phenol.

This ligase reaction mixture is subjected to transformation.

An anthranilic acid auxotrophic mutant strain LA105 (anthranilic acid synthase-deficient mutant strain) derived from Corynebacterium glutamicum strain 122 is used as a recipient bacterium to be transformed. Anthranilic acid auxotrophic mutants were obtained by selecting bacteria that cannot grow on M1 agar medium but can grow on M1 agar medium supplemented with anthranilic acid (equivalent to 30 μ/ml) through conventional mutation treatment. be done. L.A.
Preparation and transformation of protoplasts of the 105 strain were carried out in the same manner as in Example 1 (2), except that the growth medium NB was supplemented with anthranilic acid equivalent to 100 ml/ml. Transformants are selected as colonies growing on RCGP agar medium containing the equivalent of 200 Iig/ml of kanamycin. A transformed strain that can grow on Ml agar medium is obtained from the colonies that appear.

Plasmid DNA is isolated from the cultured cells of these transformed strains in the same manner as described above. Analysis of plasmid pTrp 2-3 obtained from one of the transformed strains by digestion with various restriction enzymes and agarose gel electrophoresis revealed that the 5all DNA fragment of b was inserted at approximately 7.1 into the unique 5all cleavage site of pCε53. It was found that the plasmid was

When LA105 strain was retransformed using pTrp 2-3 in the same manner, tryptophan 1100I1/
Colonies growing on RCGP agar containing ml and kanamycin 400 ttg/+nl simultaneously became anthranilic acid non-auxotrophic and they harbored a plasmid identical to pTrp 2-3 as determined by Sall's cleavage pattern. There is.

The above results indicate that approximately 7. 5al of IKb
The lDNA fragment contains Brevibacterium flavum^T.
This shows that the gene encoding CC14067 anthranilate synthase exists and is expressed in the Corynebacterium glutamicum LΔ105 strain.

The pTrp 2-3 carrying strain is American type.
Corynebacter in Cultureφ Collection
+umgglutamicum K20^TCC3903
It has been deposited as No. 5.

Plasmid pTrp 4-3 containing the gene encoding the anthranilate synthase of Brevibacterium flavum ATCC 14067 is obtained by performing the same treatment as above using Plasmid C-52.

pEC52 is a plasmid pcG1 of Corynebacterium glutamicum that the present inventors previously disclosed (Japanese patent application No. 5
6-18101 (JP 57-134500)) and Escherichia coli plasmid pG^22 [An, G, e
t 3 top: J, 13B (, teriol.

140, 400 (1979)]. For more information, see pCG There is only one Bg on I! ■There are two locations on the cleavage site and pGA22 [la
Among the mHI cleavage sites, the BamHI cleavage site in the tetracycline resistance gene is ligated using the same cohesive ends of both restriction enzymes. ρCε52 is pG^22
It has a selection marker such as the Kanamain resistance gene derived from the plant, and there is only one cleavage site for the restriction enzyme Sal I.

ρCε52 is isolated from 122 cultured strains of Corynebacterium glutamicum harboring pCε52 in the same manner as pcGll was isolated in Example 1 (1).

Similarly to the above, 36 strains of tryptophan-producing Corynebacterium glutamicum (FERM BP-451)
is transformed with pTrp 4-3. The obtained transformed strain was stored in the American Type Culture Collection as Corynebacterium glutami.
It has been deposited as cum K31° ATCC 39280.

20. Corynebacterium glutamicum carrying pTrp 2-3. ATCC39035 and pTrp4
-3 shares held 31. L- by ATCC39280
A tryptophan production test is conducted as follows.

The bacterial strain was cultured in NB liquid medium at 30°C for 16 hours with shaking.
g/i, (N)1. ) zsO* 20 g/1
,KH,PO,0,5g/I! , K2HP Exit 4
0.5 g/f -Mg5O<・7H200,2
5g/It, CaCL 20g/It, J]87
．． 2) into a test tube containing Mm and culture with shaking at 30°C for 96 hours.

After culturing, the culture p solution is subjected to paper chromatography, and after ninhydrin color development, the amount of L-)'J butophane produced is measured by colorimetric crushing.

As a control, strains LA-105 and LAII-1 are treated in the same manner.

The results are shown in Table 4.

Table 4 LA-105 Shihachi-105/pTrp 2-3 (K2O, ATCC
39035) 0.3 4LAR-10,4
8 Example 5. Made by pcBtot! ! ! :(1) p
cGll and p[lB110) separation pcG11 was obtained from Example 1 ( Isolate pCG2 in the same manner as in step 1).

ρ18110 was determined by the method of Gryczan et al.
.

T., J. et al.: J. Bacte.
ri et al., 134.318 (1978)], Bacillus subtilis BRIS'/ carrying this plasmid
pUB"' (Proc, Natl.

^cad, Sci, USA, 75.1423 (1
It is isolated from the cultured bacterial cells of 978)).

(2) In vitro recombination of pcGll and pueito pcGll plasmid DNA2J1 prepared above
Restriction fermentation containing g, iBgj! II reaction buffer (10mM
)! JS hydrochloric acid, 7mM! JgCf2.60mM
NaCl, 7mM 2-mercaptoethanol,
pH 7.5> 2 units of Bgfn (manufactured by Takara Shuzo Co., Ltd., 6 units/ρ) are added to the 100 scraps and reacted at 37°C for 60 minutes. In addition, restriction enzyme BamHI reaction buffer containing ρIIBIIO plasmid DNA 2■ (10ffllJ
) Liss hydrochloride, 7mM MgCL, 100mM NaCl
, 2mM mercaptoethanol, 0.01% bovine serum albumin, pH 8,0) 100Jd! 2 units of B
Add amHI (manufactured by Zenshuzo Co., Ltd., 6 units/〃) and heat at 37°C.
Allow to react for 60 minutes.

Mix both restriction enzyme digests and add 40% T4'J gauze buffer.
4, ATP (5mlJ) 4k, T4 ligase 0.2m
and H,0120m are added and reacted at 12°C for 16 hours. The mixture is extracted twice with phenol 4004 saturated with TES buffer to remove the phenol which is dialyzed against TBS buffer.

(3) Obtaining pcBlol Corynebacterium glutamicum LA103 was obtained in the same manner as in Example 1 (3) using 100 ρ of a 1:1 mixture of twice as concentrated TSMCjl solution and the above ligase reaction mixture as donor DNA. Transform and select kanamycin-resistant strains. Kanamycin 12.
5, icg/+nl al or spectinomycin 1
00 g/ml on NB agar medium containing 3
Three double-resistant transformants grown by culturing at 0°C for 2 days are arbitrarily selected and purified on the same agar medium. These 3 stocks are 4
Grow in 00dNB medium until OD is about 0.8,
After harvesting, plasmids are isolated from the cultured cells by ethidium bromide-cesium chloride density gradient centrifugation as described in Example 1 (1). 3 from any transformed strain.
0-35J1g of plasmid 011^ is obtained.

These plasmid DNAs were analyzed by restriction enzyme digestion and agarose gel electrophoresis in the same manner as in Example 1 (3), and the molecular weight and restriction enzymes pstl, εcoRI, HincII and Bgj! Identify the break point of II. The plasmids of the three strains all have a structure in which ρCGII and one port 8110 are harmoniously linked, and two of them have the structure shown by pcBol in FIG. 2, but the other type has the binding direction in the opposite direction.

Transformants with either plasmid are pcGll
It has spectinomycin resistance trait derived from pHB110 and kanamycin resistance trait derived from pHB110.

The kanamycin-resistant transformants obtained by re-transforming Corynebacterium glutamicum and AlO3 strains using these plasmid DNAs simultaneously acquired spectinomycin resistance and were characterized by various restriction enzyme cleavage patterns. A plasmid identical to the donor plasmid to be attached is stored.

Example 6 Corynebacterium glutamicum, Corynebacterium herculis, Brevibacterium flavum and the like were cloned using the gene involved in histidine biosynthesis of Corynebacterium glutamicum C156 strain and the expression of the gene was used. Production of histidine by Brevibacterium lactofermentum: (1) Corynebacterium glutamicum C156
Preparation of strain chromosomal DNA and plasmid pcG11: 1.
2. Corynebacterium glutamicum Cl capable of producing tehistidine resistant to 4-triazole-3-alanine
Chromosomal DNA of strain 56 (PERlJ 0P-453) is prepared in the same manner as in Example 1 (1).

On the other hand, pCG l l used as a vector plasmid
is a pcG11-bearing strain of Corynebacterium glutamicum, a derivative strain LA103 of 22 strains, and AlO3/pC.
GII (ATCC 39022) in the same manner as in Example I(1).

(2) Corynebacterium glutamicum C156
Cloning of genes involved in histidine biosynthesis of strains: Reaction solution for restriction enzyme Bgi containing pcGll plasmid DNA 3■ prepared above and chromosomal DNA 9■ above. J
gCj! 8 gl (manufactured by Zenshuzo Co., Ltd.) in lO units is added to 200 d of 2.60 mM NaCl, 7 mM 2-mercaptoethanol, and after reaction at 37°C for 60 minutes, the reaction is stopped by heating at 65°C for 10 minutes. Add T4'll gauze buffer (Tris 200mM) to this mixed digest.

MgCR266mL dithiothreitol 100ml 1J
% pH 7,6) 40ml, 40 scraps of 5mMΔTP solution, T4 ligase (manufactured by Zenshuzo Co., Ltd., 1 unit/1111) 0.
Add 3J111 and water 1201jJI and heat at 12°C.
Allow to react for 6 hours.

The T41J gauze reaction mixture is used for transformation of Corynebacterium glutamicum strain LH33 (histidine auxotrophic, lysozyme sensitive).

Transformation is performed using LH33 strain protoplasts.

Protoplasts are prepared in the same manner as in Example 1 (2).

Pour 5 ml of protoplast suspension Q into a small test tube 25
Centrifuge for 5 min at 00 × g and add 73MC buffer (I
Qm! Magnesium chloride, 30d chloride, 50
d) Squirrel, resuspended in 400mM sucrose, pH 1,5>1 ml, centrifuged and washed, then resuspended in 1 ml of TS&IC buffer Q. Add to this suspension a 1:1 mixture of twice the concentration of TSIJ [: 1:1 mixture of buffer and the above ligase-reacted DNA mixture].
Add the scraps and mix, then TSMCtj! 20 in solution
Add 3 ml of solution Q containing % PE G 6.000 and mix. After 3 minutes, add 2 m of RCGP medium (pH 7,2)
The supernatant was removed by centrifugation at 2,500xg for 5 minutes, and the precipitated protoplasts were suspended in 1ml of RCGP medium. Medium (RCGP medium containing 1.4% agar, pH 7,
21 and cultured at 30°C for 7 days.

The spectinomycin-resistant colonies grown on the selection plate were collected, and after centrifugal washing twice with physiological saline,
The mixture is spread on a minimal agar medium Ml containing 1100 A/ml of subectinomycin and cultured at 30°C for 2 days to select transformants that are resistant to svectinomycin and are not auxotrophic for histidine.

A plasmid is isolated from one of the transformed strains by ethidium bromide/cesium chloride density gradient centrifugation as described in Example 1 (1). DNA fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes are analyzed by agarose gel electrophoresis to identify the mode of restriction enzyme cleavage of this plasmid DNA. This plasmid was named pPH8. pPH8 is pCG 11 B
gI! This is a structure in which the DNA fragment b is inserted at approximately 10.6 points into the If cleavage site.

Furthermore, using pPH8DNA, strain H33 [0113
Parent strains of 3 strains (histidine auxotrophy, lysozyme resistance): F
When ERIJ 0P-4523 was re-transformed, all of the transformed strains selected as spectinomycin-resistant strains were non-requiring for histidine.

From these results, it is clear that the genes involved in histidine biosynthesis of the histidine-producing bacterium strain 0156 have been cloned.

Cloning of genes involved in histidine production can also be performed from the beginning using strain 833 as a host strain.

(3) Production of monohistidine by Corynebacterium glutamicum strain harboring pPH8 Corynebacterium glutamicum LA-103 strain (FERM
P-5947, ATCC31866) to pPH8D N
Transformed with A, spectinomycin 400 g/m
Transformants resistant to the same drug are selected on an RCGP agar medium containing L. After purifying the obtained transformant, plasmid isolation and structural analysis were performed in the same manner as above, and it was confirmed that the plasmid had the same structure as pP)13.

pPH8 carrying strain Corynebacterium glutamicum LA
103/pPH8 is Corynebacter+umg in the American Type Culture Collection
lutamicum K32. It has been deposited as ATCC39281.

Corynebacterium glutamicum and AlO3/pcG
II (ATCC39Q22) right and same [, Alf13
/pPH8 (ATCC39281) L-histidine production test is conducted as follows.

The above bacteria, cultured overnight at 30°C on NB agar medium, were cultured in 5 ml of production medium P5 (molasses 12
% (as sugar), H, Po, 0.2%, K, 1 (
Po, 0.1%, Mg5Ot・7FI2CI 0
．． 05%, NaCfO, 25%, (NH4)2SO
42.3%, urea 0.2%, CaC0z 2%, pH
7.4 (adjusted with ammonia)]. 7 at 30℃
After culturing for 5 hours, the amount of histidine produced in the medium was measured using a colorimetric method using sulfanilic acid (Pauley) reagent [H, Pau
ly.

Hoppe-Seylers; Z, Physio
lo, Chem, 42.508 (1904),
94.284 (1915)). The results are shown in Table 5.

Table 5 LA103/I) CGII OLA 10
3/pPH8(K32) 2.6(4) p
Production of histidine by Corynebacterium herculis, Brevibacterium flavum and Brevibacterium lactofermentum carrying PH8: Corynebacterium herculis^TCC13868
, Brevibacterium flavum ATCC14067 and Brevibacterium lactofermentum^TC
In order to make C13869 carry plasmid ppH8, transformation is performed using each strain as a recipient strain.

Each strain was grown in 33M medium, and the OD6601m was 0.
When the concentration reaches 2, penicillin G is added at a concentration of 0.3 units/ml. Cultivation is continued, and when O[1660 nm increases to 0.6, bacteria are harvested, and protoplasts are formed in the same manner as described above in RCGP medium containing 1 mg/ml lysozyme. Transformation is performed using pPHg according to the method described above, and the transformed strain is selected as a colony growing on an RCGP agar medium containing 400 μl/ml of spectinomycin.

Plasmid DNA was obtained from the cultured cells of the purified subectinomycin-resistant transformant strain in JP-A-57-183799.
57-134500, and it was confirmed by restriction enzyme cleavage that they have the same structure as pPHg. From the above, plasmid ρCGII
Plasmid pPH8, which is a derivative of , can be replicated in Corynebacterium herkyulis, Brevibacterium flavum and Brevibacterium lactofermentum, indicating that plasmid pcGll can be used widely in these bacterial species.

33 for Corynebacterium herculis, 34 for Brevibacterium flavum, and 35 for Brevibacterium lactofermentum, which are strains carrying ppH8, were sent to the American Type Culture Collection at ATCC 39282, 39283, and 392, respectively.
It has been deposited as No. 84.

L-histidine production tests using these strains are conducted as follows.

One platinum loop of each pPH8-carrying strain and its parent strain, cultured overnight at 30° C. on NB agar medium, is inoculated into 5 ml of production medium P5. After shaking culture at 30°C for 75 hours,
L-histidine production in the medium is determined colorimetrically by the Portu method. The results are shown in Table 6.

Table 6 From the above table, genes involved in histidine production derived from Corynebacterium glutamicum are derived from Corynebacterium glutamicum.
In addition to Glutamicum, it was expressed in Corynebacterium herculis, Brevibacterium flavum, and Brevibacterium lactofermentum, and it was clear that it contributed to the production of histidine.

[Brief explanation of the drawing]

FIG. 1 shows the restriction enzyme map of plasmid pG112. Figure 2 shows the restriction enzyme map of plasmid pcB1 old. FIG. 3 shows a flowchart for the construction of plasmid p6thrl. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. ^TCC1
3869/pPH8 (35.ATCC39284)2
．． 0 Figure ■ colu 1゜eye name+11amllI/F3glH

Claims (1)

[Claims] A microorganism that belongs to the genus Corynebacterium or Brevibacterium and is derived from a species of the genus Corynebacterium or Brevibacterium and is sensitive to S-(2-aminoethyl)-cysteine is referred to as S-(2-aminoethyl)- DNA containing a gene that has the activity of converting into a cysteine-resistant strain
A microorganism carrying a recombinant DNA of the fragment and a vector DNA capable of autonomous replication in Corynebacterium or Brevibacterium species is cultured in a culture medium, and L
- A method for producing L-lysine, which comprises producing and accumulating lysine and collecting L-lysine from the culture.